Methods for identifying and isolating unique nucleic acid sequences

Abstract

A subtractive suppression hybridization (SSH) assay and uses thereof are described. In particular, methods of identifying and isolating nucleic acid sequences, which are unique for a certain cell, tissue or organism are provided, wherein said unique nucleid acid sequences are related to for example diseases genes. More specifically, SSH assays for unique genomic DNA sequences and improved SSH assays that are combined with 2D gel electrophoresis techniques are provided. The presented methods are particular useful for the identification of genes involved in the development of various diseases, including cancer, hypertension and diabetes as well as for monitoring animals and food, for example for infection agents and other contaminants.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a subtractive suppression hybridization (SSH) assay and uses thereof. In particular, the present invention relates to methods of identifying and isolating nucleic acid sequences, which are unique for a certain cell, tissue or organism, wherein said unique nucleid acid sequences are preferably related to genes that are etiologically related to a disease. More specifically, the present invention is directed to SSH assays for unique genomic DNA sequences and to improved SSH assays that are combined with 2D gel electrophoresis techniques. The presented methods are particular useful for the identification of novel genes involved in the development of various diseases, including cancer, hypertension and diabetes as well as for monitoring animals and food, for example for infection agents and other contaminants. BACKGROUND ART [0002] Currently, there are two basic approaches to determine the relationship between abnormal genes ...

AI Technical Summary

Benefits of technology

[0014] The invention includes the rapid enrichment of differences between two organisms and is a substantial improvement of a technique published by Diatchenko, et al. (1999). Suppression subtractive hybridisation (SSH) is a cost-effective and powerful technique for the isolation of species-specific DNA sequences from closely related microorganisms. The principle behind this technique is a two-step hybridization with an excess of genomic DNA from a “driver” organism compared with that from a “tester” organism. After reannealing tester and driver DNA, only specific DNA fragments (tester DNA) with an appropriate pairs of adapter can participate in an exponential PCR amplification when defined oligonucleotide is used as primer. Next, a secondary PCR amplification is performed using nested primers to further reduce background PCR products and enrich for tester-specific sequences.

[0024] The methods of the present invention can primarily be used to compare genomic sequences, preferably of eukaryotic origin, from different cells / tissues / organisms with the purpose of identifying unique gene sequences. Furthermore, the improved methods of the present invention described herein can be used to identify differences in gene expression from cells / tissues / organisms using, RNA and / or cDNA. In addition, the methods of the present invention can be used to identify minute differences between similar organisms such as infectious agents to obtain sequence data of antibiotic resistance Genes for improving the efficaciousness of treatments. In an important aspect of the instant invention the disclosed methods can be used to identify differences in two different genomes which are very closely related to obtain genes involved in the development of various diseases, including cancer, hypertension, and diabetes as well as differences in gene expression that are relevant to disease and / or caused by exposure to toxicants.

[0037] As described in the examples, a new combined technique is provided comprising SSH and a specific two dimensional polyacrylamide gel electrophoresis that reduces the unspecific PCR fragment background even when lower eukaryotes such as the mold P. nalgiovense are analysed. Because the specific tester DNA fragments were demarcated from the background it was possible to directly identify the fragments by sequencing.

Problems solved by technology

The availability of these genes for mutational analyses is, however, very limited.

Due to the large number of candidate genes, experimental variations and availability of appropriate software become the limiting factors in associating, certain expression patterns with disease.

Several methods have been used to distinguish differences between two organisms or two genomes, but have not been used in identification of disease genes.

One cannot assure the exact amplification of the tester and driver DNA used for further comparison.

Therefore, differences introduced by the initiated PCR will consequently lead to artificial differences after hybridization.

This method is found to be very time consuming and not suitable for routine analysis (Lisitsyn et al.

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